Desmoplakin (DP) is the most abundant component of desmosomes, intercellular junctions required for maintaining tissue integrity in embryos and adults. Loss of DP results in early embryonic lethality in mice, and DP mutations in humans cause skin and heart defects. Work performed during the last funding period supports our hypothesis that DP forms a multi-functional scaffold where differentiation-specific components converge to couple intermediate filaments (IF) to the membrane. Based on our live cell imaging analysis, we now propose that the proper construction of the DP-IF scaffold requires that interaction among DP and its partners be precisely coordinated spatially and temporally during junction assembly. In the next period we will continue to elucidate critical structural features of the DP-IF scaffolding, to investigate the molecular and regulatory mechanisms driving its assembly, and to determine how defects in assembly due to engineered or naturally occurring DP human mutations lead to structural and functional defects in vitro and in vivo. Our specific goals are: 1. To determine how sequences within the DP C-terminus specify interactions with different IF by testing the effects of engineered and naturally occurring DP mutations in vitro and in cells on binding to cell type-specific IF;2. To determine how DP collaborates with the armadillo family member PKP2 in assembly and regulation of the DP-IF scaffold by a) investigating the biochemical, temporal and spatial relationship of DP and PKP2 during junction assembly, b) identifying DP or PKP2 mutants that uncouple DP-PKP2 or PKP2-actin, and c) examining effects of these mutants and RNAi-mediated PKP2 knock down on desmosome assembly and function;3. To investigate how intracellular calcium stores and downstream signaling regulate DP trafficking to desmosomes by analyzing DP assembly behavior in keratinocytes in which ATP2A2/SERCA2 is impaired pharmacologically or by Darier's disease mutations or RNAi-mediated knock down;and 4. To define how engineered and naturally occurring DP mutations that regulate DP-IF interactions affect desmosome assembly and maturation, adhesive strength and wound healing in vitro and in an animal model. These experiments will help us to understand how DP mutations and defects in desmosome assembly and structure lead to human skin disease, and also to appreciate why carefully regulating desmosome assembly is important for proper epidermal wound healing.